OBJECTIVES: (1) We intend to investigate the interactions of purified 32P labelled spectrin tetramers with spectrin depleted inverted red cell vesicles. All previous binding studies have used spectrin heterodimers, (Bennett and Branton, 1977, Yu and Goodman, 1979), but now that there is suggestive evidence that the tetramer is the physiologically relevant unit of spectrin on the membrane it becomes essential to study the binding using the tetramer. Scatchard Plots of these binding studies should yield the definitive answer on the stoichiometry of the Spectrin-Band 2.1 interaction on the membrane. (2) We intend to study the interactions of purifed I125 labelled Band 2.1 and 4.1 with spectrin tetramers using rate zonal sedimentation through sucrose gradients for binding analysis. Bands 2.1 or 4.1 will be selectively cleaved enzymatically or chemically into fragments separable by gel filtration. Fragments will be (a) tested for the ability to bind spectrin, (b) two dimensional peptide maps will be derived (Yu and Goodman), and (c) n terminal and c terminal analysis performed. In this manner we hope to order the fragments within the protein and identify the binding sites. (3) The same approach will be used to ask whether purified Band 2.1 can bind to purified Band 3 in solution, and if so to dissect these proteins individually in search of their active binding sites. (4) The proteins described above will also be purified from abnormally shaped hereditary spherocytes (HS) and hereditary elliptocytes (HE). In both hemolytic anemias it has been suggested that the primary defect resides either within the spectrin cytoskeletal network or the attachment of this network to the membrane (Lux, 1979). We hope to define the exact molecular defects(s) which cause these red cell abnormalities and enhance our knowledge of the essential protein interactions in normal cells by asking the questions outlined in 1-3 making use of purified proteins derived from HS and HE erythrocytes.